The major aim of this research proposal is to elucidate the mechanism of synthesis of Semliki Forest virus (SFV) 26S mRNA and the processing in vitro of its translation products. We intend to determine whether synthesis of this RNA initiates at an internal site of its template, the 42S minus strand RNA, or whether synthesis initiates at the 3'-ter-minus of the template followed either by a "stop-start" progression of the polymerase or by cleavage and processing of the entire 42S RNA transcript. (1) We will determine the target size of its template by u.v. irradiation of infected cells and the SFV replication complex which synthesizes this RNA in vitro. (2) We will characterize the 5'-termini of in vivo and in vitro synthesized 26S mRNA's. (3) We will determine the sequence of about 80 nucleotides at the 5'-termini of both 26S mRNA and 42S virion RNA. About 50% of RF's and RI's found in infected cells can be cleaved by low levels of RNase A to yield two cores, RFII and RFIII. This cleavage results in loss of from 300 to 2000 nucleotides of the sequence in minus strand RNA unprotected by plus strands. We will determine the nature of the "gap region" in RF's and RI's by identifying the oligo- and monocleotides released by treatment with nuclease. We will fill in this gap between plus strands of RF's and RI's with DNA complementary to part or all of the unprotected sequence in minus strand RNA using reverse transcriptase, and we will sequence this cDNA. We will continue in our purification and characterization of the SFV RNA polymerase to identify components necessary for synthesis of all SFV RNA's. Finally, we will establish an in vitro protein-synthesizing system which includes membranous vesicles from dog pancreas. The mRNA for this system will be 26S mRNA. We will identify which proteins are released free and which are sequestered in membranes. With the use of appropriate mutants, we will identify whether there is one signal sequence or more than one controlling association with membranes of the two glycoproteins synthesized in vitro.